The following includes information that may be useful in understanding the present inventions. It is not an admission that any of the information provided herein is prior art, or material, to the presently described or claimed inventions, or that any publication or document that is specifically or implicitly referenced is prior art.
The present invention relates to, but is not limited to, the fields of microbiology and microbial genetics. The invention relates, for example, to novel bacterial strains, novel nucleotide sequences, novel amino acid sequences, and processes for employing these bacterial strains, novel nucleotide sequences, and/or novel amino acid sequences for fermentative production of amino acids including, but not limited to, L-threonine, L-lysine, L-homoserine, and L-isoleucine. Preferably, L-threonine is produced. The invention also relates to the production of animal feed additives. The invention also relates to fermentation and synthesis of fine chemicals including but not limited to those amino acids listed above.
In Escherichia coli, the amino acids L-threonine, L-isoleucine, L-homoserine, L-lysine and L-methionine derive all or part of their carbon atoms from aspartate (aspartic acid) via a common biosynthetic pathway (G. N. Cohen, “The Common Pathway to Lysine, Methionine and Threonine,” pp. 147-171 in Amino Acids: Biosynthesis and Genetic Regulation, K. M. Herrmann and R. L. Somerville, eds., Addison-Wesley Publishing Co., Inc., Reading, Mass. (1983)):

Aspartate is, in turn, derived from oxaloacetate (OAA). As reported in U.S. Pat. No. 6,455,284, to Gokarn, et al., aerobic fermentation can be used to produce oxaloacetate-derived amino acids. Unfortunately, process yields may be limited by stringent metabolic regulation of carbon flow. In general, carbon flux toward OAA is said to remain constant regardless of perturbations to the metabolic system. J. Vallino, et al., Biotechnol. Bioeng., 41: 633, 646 (1993). Overcoming this metabolic regulation would be advantageous in increasing production of OAA-derived amino acids and other products.
In aerobic bacterial metabolism, carbon atoms of glucose may be fully oxidized to carbon dioxide in the tricarboxylic acid cycle (TCA), also known as the citric acid or Krebs cycle. The TCA cycle begins when OAA combines with acetyl-CoA to form citrate. An example of the aerobic metabolism pathway in the bacterium Escherichia coli is shown in FIG. 1. In addition to its role as a primary molecule in the TCA cycle, OAA may be used as a precursor for synthesis of amino acids, including L-asparagine, L-aspartate, L-methionine, L-threonine, L-isoleucine, L-homoserine, and L-lysine.
Given the importance of OAA to the TCA cycle, OAA that is used for biosynthesis of amino acids should be replaced to allow further progress of the TCA cycle. Many organisms have therefore developed “anaplerotic pathways” that regenerate intermediates for use in the TCA cycle. In some organisms, for instance in some plants and microorganisms, TCA cycle intermediates may be formed from acetyl-CoA via an anaplerotic pathway known as the “glyoxylate shunt,” also known as the “glyoxylate bypass” or “glyoxylate cycle.” The glyoxylate shunt in E. coli is shown in FIG. 2.
The glyoxylate shunt allows organisms growing on certain substrates (for instance, acetate, fatty acids, or some long-chain alkanes) to replenish their OAA. Such a mechanism is useful because such substrates do not provide 3-carbon intermediates that can be carboxylated to form OAA needed in the TCA cycle. The branch point of carbon flux between the TCA cycle and the glyoxylate shunt is said to be isocitrate (K. Walsh et al., J. Biol. Chem. 259:15, 9646-9654 (1984)).
In the glyoxylate shunt, isocitrate from the TCA cycle is cleaved into glyoxylate and succinate by the enzyme isocitrate lyase. The enzyme malate synthase is then used to combine glyoxylate with acetyl-CoA to form malate. Both succinate and malate may be used to generate OAA through the TCA cycle. In general, expression of genes encoding the glyoxylate bypass enzymes is said to be rigidly controlled, such that these genes may be repressed when certain 3-carbon compounds are available for use in the TCA cycle.
The following reactions may be observed:aceA, isocitrate lyase: isocitrate <--> glyoxylate+succinateaceB, malate synthase A: acetyl-CoA+H2O+glycoxylate <--> malate+CoAglcB, malate synthase G: acetyl-CoA+H2O+glycoxylate <--> malate+CoA
In E. coli, genes encoding glyoxylate shunt enzymes are located in the aceBAK operon. They are said to be controlled by a number of transcriptional regulators including, for instance, IclR (A. Sunnarborg et al., J. Bact., 172: 2642-2649 (1990)), FadR (S. Maloy et al., J. Bact. 148: 83-90 (1981)), FruR (A. Chia et al., J. Bact., 171: 2424-2434 (1989)), and ArcAB (S. Iuchi et al., J. Bact., 171: 868-873 (1989)).
The aceBAK operon has been reported to be expressed from a σ70-type promoter that is upstream of aceB (E. Resnik et al., J. Bact. 178:9, 2715-2717 (1996)). A nucleotide sequence for the aceBAK operon of the E. coli strain K-12 is set forth in SEQ ID NO:1. Keseler, I. M., et al., Nuc. Acids Res., 33: D334-357 (2005). The operon is said to be regulated by a repressor protein expressed from iclR and activated by growth on acetate or fatty acids (E. Resnik et al., supra.). The aceA gene (SEQ ID NO:2) (Keseler, I. M., et al., supra) is reported to encode isocitrate lyase (SEQ ID NO:3) (Keseler, I. M., et al., supra), and the aceB gene (SEQ ID NO:4) (Keseler, I. M., et al., supra) is reported to produce malate synthase A (SEQ ID NO:5) (Keseler, I. M., et al., supra). The final gene in the glcDFGB operon (SEQ ID NO:6) (Keseler, I. M., et al., supra), glcB (SEQ ID NO:7) (Keseler, I. M., et al., supra), is reported to encode malate synthase G (SEQ ID NO:8) (Keseler, I. M., et al., supra), which may replace malate synthase A in the glyoxylate shunt when malate synthase A is absent. (L. N. Omston, et al., J. Bact., 98:2, 1098-1108 (1969); W. Farmer, et al., App. & Env. Microbiol., 63:8, 3205-3210 (1997); M. Oh, et al., J. Biol. Chem., 277:15, 13175-13183 (2002).)
Many features of wild-type strains of E. coli have been reported. For instance, the genome of E. coli strain K-12 is reported in F. R. Blattner, et al., Science, 1997 Sep. 5; 277(5331): 1453-74. Some authors have reported attempts to divert carbon flow toward OAA, since it was postulated that increasing flow of carbon toward OAA would increase production of biochemicals that may be synthesized with OAA as a precursor. Efforts have included either knockout of genes that act as aceBAK repressors or enhancement of genes that inhibit aceBAK (to avoid carbon flow into the glyoxylate shunt). For instance, U.S. Pat. No. 6,630,332, to Rieping et al., reports increased threonine production in Enterobacteriaceae through over-expression of the mqo gene, which produces the enzyme malate:quinone oxidoreductase.
European Patent Application No. EP 1 408 123 A1, to Park, et al., reports production of L-threonine using a microorganism in which the fadR gene has been knocked-out. U.S. Patent Application Publication No. 2003/0059903A1, to Rieping, et al., and International Publication WO 02/081722, to Rieping, et al., report a process for the production of L-threonine including fermentation of Enterobacteriaceae in which the aceA gene or nucleotide sequences encoding for the aceA gene are attenuated or switched off.
International Patent Publication No. WO 03/038106A2, to Rieping, et al., reports a process for production of L-threonine using bacteria modified to enhance activity levels of the fadR gene product and/or the iclR gene product, both of which are transcriptional repressors of the aceBAK operon. International Patent Publication No. WO 03/008616, to Hermann, reports a process for the preparation of L-threonine including fermentation of bacteria of the Enterobacteriaceae family that have been modified so that the expression of the aceK gene product is attenuated.
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Each of the foregoing references, and those in the description that follows, are incorporated herein by reference to the extent necessary to aid one of ordinary skill in the art to understand or practice the further teachings provided by the present disclosure.
There remains a need in the art for microorganism strains that are culturable and produce increased amounts of amino acids such as L-threonine, L-methionine, L-lysine, L-homoserine, and L-isoleucine.